1. Field of the Invention
The invention pertains to the field of experimental animals. More particularly, the invention pertains to experimental animals suffering corneal epithelial damages, such as dry eye, and methods of using the same to assay a variety of compounds for evaluating the therapeutic effect thereof on said disease.
2. Description of Related Art
The cornea is a transparent tissue of 0.52 to 1.0 mm thick, and constitutes together with the sclera an important barrier to foreign substances, and is organized into five layers: epithelium, Bowman's membrane, stroma, Descemet's membrane and endothelium.
The term “corneal epithelial damage” usually refers to a group of diseases in which the epithelium cells of the corneal epithelial layer, the forefront segments of the cornea, are partly or wholly impaired, damaged and/or abraded. Depending on the presence or absence of ulcers in the basement membrane or corneal stroma, the symptoms of these diseases are classified into superficial punctate keratopathy (SPK), simple corneal erosion, recurrent corneal erosion, and corneal abrasion. The diseases may be caused by lacrimal fluid disorders, diseases of eyelids and/or conjunctiva, metabolic disorders, infections, trauma, drugs, and the like. Among them, dry eye, which is caused by lacrimal fluid disorders, has been drawing much attention in recent years.
The term “dry eye” includes a group of diseases, which occur on the ocular surface, caused by the loss of lacrimal fluid.
Lacrimal fluid plays an important role in helping an ocular function normally. For example, 1) it keeps keratoconjunctiva moist or protects it from being dried; 2) the lacrimal fluid layer keeps the surface of the cornea smooth and functions as a transparent optical refractive medium; 3) antibacterial ingredients in lacrimal fluid prevent viral infection, bacterial infection and so on; 4) it flushes out foreign substances and/or wastes adhered to the surface of the keratoconjunctiva; 5) it supplies oxygen, water and nutrients to the cornea, one of the few tissues having no blood vessel therein; 6) it makes the blinking or ocular movement smooth; and 7) it functions as a migration path for leukocytes in cases of ocular trauma.
The surface of the eyeball in a healthy condition is worn by lacrimal fluid. A layer of new lacrimal fluid (lacrimal fluid layer) is formed at every blinking, so that the ocular surface is protected from drying. While this lacrimal fluid layer begins to dry in approximately ten seconds, the sensory nerve distributed over the ocular surface detects drying and induces blinking, thereby keeping the ocular surface always moistened with lacrimal fluid. This mechanism is known as a basal secretion of lacrimal fluid, and is distinguished from a reflex secretion, which occurs when sad or when dust gets into the eye. The surface of the keratoconjunctiva will get dry and be damaged if the volume of lacrimal fluid decreases.
Dry eye has been called an ocular xerosis or a hypolacrimia, which for the most part has been considered as one symptom of Sjogren's syndrome. However, types of dry eye, which impair only the basal secretion without Sjogren's syndrome, due to the use of VDT (visual display terminal, or simply “display”), are increasing in recent years.
In a visual information society of these days, the number of VDT workers is increasing rapidly in parallel with the growing use of OA apparatus including computers. The total number of dry eye patients in Japan now is estimated to be from one million to two million, and the number of patients is still increasing, including potential dry eye patients.
The frequency of blinking will decrease when people continue watching the display (VDT) of OA apparatus for a long hour. Since lacrimal fluid is supplied to the ocular surface while the eye is closed by blinking, said decrease in the frequency of blinking leads to reduction in volume of lacrimal fluid, and is causative of corneal damages. It is expected therefore that the number of dry eye patients will continue to increase due to the heavy burden to be imposed on eyes.
Besides the decrease in said blinking frequency brought about by the above VDT work, endogenous diseases, such as Sjogren's syndrome, dry keratoconjunctivitis and Stevens-Johnson syndrome, are causative of dry eye. Cataract operation, medicines, trauma, contact lenses and others may also be causative of dry eye, due to the quantitative or qualitative deterioration of lacrimal fluid.
Various medicines for treatment of the corneal epithelial damages, such as dry eye, have been made available on the market. However, the therapeutic effects of these medicines are unsatisfactory to meet with medical needs, and R & D of new medicines is extensively in progress. Such R & D of new medicines cannot be accomplished without suitable systems or model animals for evaluating the effect of each test compound on the corneal epithelial damages.
Known experimental model animals suffering corneal epithelial damage include a rat Vitamin A-deficient model (Japan Patent Publication No. H09-136832). In this model, a dry eye-like symptom is produced by breeding rats under Vitamin A-deficient conditions, and is healed by giving Vitamin A to said rats (oral or eye drop administration). Thus, these malnutritional model animals are still unsuitable to evaluate medicines having various mechanisms of action.
Other known model animals in this field include those whose corneal epithelial damages are produced by treatment with iodine gas or an organic solvent (e.g., n-heptanol); by physical treatment with, for example, a knife; or by alkali corrosion treatment using, for example, sodium hydroxide. Like dry eye, these model animals suffer corneal epithelial damages; however, said damages produced by the above-mentioned methods are healed within a short period of time and are inconvenient to evaluate the therapeutic effects of medicines. Thus, the development of model animals having prolonged corneal epithelial damages, which resist various medical treatment, have long been desired. (See Teruo Nishida “Development of Medicine; vol. 9; Search for Medicine I”, pp. 287-291, published by Hirokawa Shoten, Tokyo, Japan (1990)).
Further known model animals in this field include a rabbit model which is produced by the compulsive eyelid retraction method (rabbit compulsive eyelid retraction model) (Kei Nagano et al., New Ophthalmology, 13 (2), pp. 267-270(1996)). In this model, the upper and lower eyelids of both eyes are everted under general anesthesia, are sutured, and are retracted compulsively for three hours. Said air-drying step deprives lacrimal fluid of water on the ocular surface, resulting in decrease in the water content of the ocular tissues, and the model animals thus obtained show dry eye-like symptoms. As seen from the above, this model animal is prepared by mechanisms similar to “dry eye” itself and, as compared with other model animals mentioned above (e.g., rat Vitamin A-deficient model), is more appropriate to be used for the assay of medicines having various mechanisms of action.
This model animal, however, is still unsatisfactory in that 1) it takes a long time, about 3 hours, to produce the model; 2) it is hard to steadily produce model animals having a predetermined damage on the surface of the cornea; 3) the corneal damage is not uniform on its surface; and 4) said damage is not maintained for a period of time sufficient to evaluate the therapeutic effect of medicines, as it is healed easily when moistened with lacrimal fluid by blinking.